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RayBio Label-Based (L-Series) Human Antibody Array 507 (L-507) or 493 (L-493) Patent Pending Technology User Manual (Re...
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RayBio Label-Based (L-Series) Human Antibody Array 507 (L-507) or 493 (L-493) Patent Pending Technology User Manual (Revised September 9, 2014) For the simultaneous detection of the relative expression of 507 (L-507) or 493 (L-493) human proteins in serum, plasma, cell culture supernatants, cell/tissue lysates or other body fluids.

L-Series Human Antibody Array L-507 Cat# AAH-BLG-1-2 (2 Sample Kit) Cat# AAH-BLG-1-4 (4 Sample Kit) L-Series Human Antibody Array L-493 Cat# AAH-BLG-2-2 (2 Sample Kit) Cat# AAH-BLG-2-4 (4 Sample Kit)

Please read manual carefully before starting experiment

Your Provider of Excellent Protein Array Systems and Services Tel: (Toll Free) 1-888-494-8555 or +1-770-729-2992; Fax: +1-770-206-2393; Website: www.raybiotech.com Email: [email protected]

TABLE OF CONTENTS I. II.

Introduction and How It Works…………………………………………………………………………………2 Materials Provided…………………………………………………………………………………………………………………… 3 A. Storage Recommendations…………………………………………………………………………………… 3 B. Additional Materials Required……………………………………………………………………..…… 3 III. Overview and General Considerations……………………………………………….…………… 4 A. Preparation and Storage of Samples………………………………………………………… 4 B. Handling the Glass Slides…………………………………………………………………………………….…… 6 C. Layout of Human L-507 or L-493 Slide…………………………………………..……… 7 D. Incubation and Washes……………………………………………………………………………………………… 7 IV. Protocol………………………………………………………………………………………………………………………………………..………… 8 A. Dialysis of Sample……………………………………………………………………………………………………………… 8 B. Biotin Labeling of Sample…………………………………………………………………………………………9 C. Drying of the Glass Slide…………………………………………………………………………………………… 11 D. Blocking and Incubations……………………………………………………………………………….…………11 E. Fluorescence Detection…………………………………………………………………………………..………… 14 V. Antibody Array Maps and Target Lists…………………………………………………..………… 15 A. RayBio Human Antibody Array L-507 Map………………………………………… 15 B. RayBio Human Antibody Array L-507 Target List…………………….… 16 C. RayBio Human Antibody Array L-493 Map………………………………………… 18 D. RayBio Human Antibody Array L-493 Target List……………….……… 19 VI. Interpretation of Results…………………………………………………………………………………………………… 21 A. Explanation of Controls Spots…………………………………………………………………….……… 21 B. Typical Results……………………………………………………………………………………………………………….……… 21 C. Background Subtraction……………………………………………………………………………………….……23 D. Normalization of Array Data…………………………………………………………………………………23 E. Threshold of Significant Difference…………………………………………………………..… 24 VII. Troubleshooting Guide………………………………………………………………………………………………..……… 25 VIII. Selected References……………………………………………………………………………………………………..………… 26 RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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I. Introduction Recent technological advances by RayBiotech have enabled the largest commercially available antibody array to date. With the L-Series Antibody Array 507 or L-493, researchers can now obtain a broad, panoramic view of cytokine expression. The expression levels of 507 or 493 human target proteins can be simultaneously detected, including cytokines, chemokines, adipokines, growth factors, angiogenic factors, proteases, soluble receptors, soluble adhesion molecules and other proteins in cell culture supernatants, serum and plasma. The first step in using the RayBio® L-Series Human Antibody Array 507 or L493 is to biotinylate the primary amine groups of the proteins in your sample (sera or plasma, cell culture supernatants, cell lysates or tissue lysates). The glass slide arrays are then blocked, just like a Western blot, and the biotin-labeled sample is added onto the glass slide, which is pre-printed with capture antibodies. The slide is incubated to allow binding of target proteins. Streptavidin-conjugated fluorescent dye (Cy3 equivalent) is then applied to the array. Finally, the glass slide is dried, and laser fluorescence scanning is used to visualize the signals.

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II. Materials Provided A. Storage Recommendations Upon receipt, the kit should be stored at -20°C until needed. Use within 6 months from the date of shipment is recommended. After initial use, remaining reagents should be stored at 4°C and may be stored for up to 3 months (Labeling Reagent, Item B, should be prepared fresh each time before use). Unused glass slides should be kept at -20 °C and repeated freeze-thaw cycles should be avoided (slides may be stored for 6 months). DESCRIPTION ITEM

NOTE: all components (except slides) are identical between the L-493 and L-507 kits

A B D

Dialysis Vials & Floating Dialysis Rack Labeling Reagent Stop Solution RayBio® L-Series Human Antibody Array L-507 or L-493 Glass Slides* Blocking Buffer 20X Wash Buffer I 20X Wash Buffer II Cy3-Conjugated Streptavidin** Adhesive Plastic Strips Labeling Buffer 2X Cell Lysis Buffer*** 30 ml Centrifuge Tube

E F G H I J K n/a M

AAH-BLG-1-2 (L-507) or AAH-BLG-2-2 (L-493)

AAH-BLG-1-4 (L-507) or AAH-BLG-2-4 (L-493)

4 vials 1 vial

8 vials 2 vials

1 vial (50 µl) 1 slide (L-507 or L2 slides (L-507 or L493) 493) 1 bottle (8 ml) 1 bottle (8 ml) 1 bottle (30 ml) 1 bottle (30 ml) 1 bottle (30 ml) 1 bottle (30 ml) 1 vial 2 vials 1 bottle (8 ml) 1 bottle (10 ml) 1 tube

*Each slide contains 2 identical subarrays **HiLyte PlusTM 555 ***Only needed if testing cell or tissue lysates

B. Additional Materials Required • • • • • • •

KCl, NaCl, KH2PO4, Na2HPO4 and ddH2O 1 ml tube, small plastic or glass containers Orbital shaker or oscillating rocker Beaker, stir plate and stir bar Pipettors, pipette tips and other common lab consumables Laser scanner for fluorescence detection (list available online) Aluminum foil

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III. Overview and General Considerations A. Preparation and Storage of Samples 1) Preparation of Cell Culture Supernatants 1. Seed cells at a density of 1x106 cells in 100 mm tissue culture dishes.* 2. Culture cells in complete culture medium for ~24–48 hours.** 3. Replenish with serum-free or low-serum medium such as 0.2% FCS/FBS serum, and then incubate cells again for ~48 hours.**,† The membrane-based array is recommended if high serum medium such as 10% FCS/FBS is used, as high background can occur on glass slide arrays with high serum containing media samples. 4. To collect supernatants, centrifuge at 1,000 g for 10 min and store as ≤1 ml aliquots at -80°C until needed. 5. Measure the total wet weight of cultured cells in the pellet and/or culture dish. You may then normalize between arrays by dividing fluorescent signals by total cell mass (i.e., express results as the relative amount of protein expressed/mg total cell mass). Or you can normalize between arrays by determining cell lysate concentration using a total protein assay (BCA Protein Assay Kit, Pierce, Prod #: 23227). *The density of cells per dish used is dependent on the cell type. More or less cells may be required. **Optimal culture time may vary and will depend on the cell line, treatment conditions and other factors. †Bovine serum proteins produce detectable signals on the RayBio® LSeries Array in media containing serum concentrations as low as 0.2%. When testing serum-containing media, we strongly recommend testing an uncultured media blank for comparison with sample results. RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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2) Extracting Protein from Cells 1. Centrifuge Cells: a. Adherent Cells: i. Remove supernatant from cell culture and wash cells gently twice with cold 1X PBS taking care not to disturb cell layer. ii. Add enough cold 1X PBS to cover cell layer and use cell scraper to detach cells. Proceed to b. Cells in Suspension. b. Cells in Suspension: Pellet the cells by centrifuging using a microcentrifuge at 1500 rpm for 10 min. 2. Make sure to remove any remaining PBS before adding 1X Cell Lysis Buffer (2X Cell Lysis Buffer should be diluted 2 fold with ddH2O). Solubilize the cells at 2x107 cells/ml in 1X Cell Lysis Buffer. 3. Pipette up and down to resuspend cells and rock the lysates gently at 2–8 °C for 30 minutes. Transfer extracts to microfuge tubes and centrifuge at 13,000 rpm for 10 min at 2-8 °C. Note: If the lysates appear to be cloudy, transfer the lysates to a clean tube, centrifuge again at 13,000 rpm for 20 minutes at 2-8°C. If the lysates are still not clear, store them at -20°C for 20 minutes. Remove from the freezer and immediately centrifuge at 13,000 rpm for 20 minutes at 28°C. 4. Transfer lysates to a clean tube. Determining cell lysate concentrations using a total protein assay (BCA Protein Assay Kit, Pierce, Prod# 23227). Aliquot the lysates and store at -80°C. 3) Extracting Protein from Crude Tissue 1. Transfer approximate 100 mg crude tissue into a tube with 1 ml 1X Cell Lysis Buffer (2X Cell Lysis Buffer should be diluted 2 fold with ddH2O). RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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2. Homogenize the tissue according to homogenizer manufacturer instructions. 3. Transfer extracts to microcentrifuge tubes and centrifuge for 20 min at 13,000 rpm (4°C). Note: If the supernatant appears to be cloudy, transfer the supernatants to a clean tube, centrifuge again at 13,000 rpm for 20 minutes at 2-8°C. If the supernatant is still not clear, store the lysate at -20°C for 20 minutes. Remove from the freezer, immediately centrifuge at 13,000 rpm for 20 minutes at 2-8°C. 4. Transfer supernatant to a clean tube and store at -80°C. B. Handling the Glass Slides • The microarray slides are delicate. Please do not touch the array surface with pipette tips, forceps or your fingers. Hold the slides by the edges only. • Handle the slides with powder-free gloves and in a clean environment. • Do not remove the glass slide from the chamber assembly until step 20 on page 13, and take great care not to break the glass slide when doing so.

• Remove reagents/sample by gently applying suction with a pipette to corners of each chamber. Do not touch the printed area of the array, only the sides as seen in image below.

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C. Layout of Human L-507 and L-493 Glass Slide Two identical sub-arrays on one slide

D. Incubations and Washes • Cover incubation chamber with a Plastic Adhesive Strip (Item J) to prevent evaporation during incubation or wash steps, particularly those steps lasting 2 hours or longer. • During incubation and wash steps avoid foaming and remove all bubbles from the sub-array surface. • Perform all incubation and wash steps under gentle rotation or rocking motion (~0.5 to 1 cycle/sec). • Wash steps in Wash Buffer II and all incubation steps may be performed overnight at 4°C. • Avoid cross-contamination of samples to neighboring wells. To remove Wash Buffers and other reagents from chamber wells, you may invert the Glass Slide Assembly to decant, and aspirate the remaining liquid. • Unlike most Cy3 fluors, the HiLyte Plus™ 555 used in this kit is very stable at room temperature (RT) and resistant to photobleaching on the hybridized glass slides. However, please protect glass slides from directly strong light and temperatures above RT. RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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IV. Protocol Assay Diagram 1. Cell culture supernatants or cell/tissue lysates

2. Serum or plasma

Note: If using cell or tissue lysates, start at “Dialysis of sample” A. Dialysis of Sample Note: Samples must be dialyzed prior to biotin-labeling (Steps 5–7). 1. To prepare dialysis buffer (1X PBS, pH=8.0), dissolve 0.6 g KCl, 24 g NaCl, 0.6 g KH2PO4 and 3.45 g Na2HPO4 in 2500 ml ddH2O. Adjust pH=8.0 with 1M NaOH and adjust final volume to 3000 ml with ddH2O. 2. Add each sample into a separate Dialysis Tube (Item A). Loading volumes are as follows: 200 μl cell culture supernatant; 100 μl cell or tissue lysate (1~2 mg/ml total protein); 20 μl serum or plasma + 80 μl 1X PBS, pH=8 (5-fold dilution). Carefully place Dialysis Tubes into Floating Dialysis Rack.

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Note for cell culture supernatants: if using a 2-fold dilution of biotin-labeled sample in the array incubation step (page 11, step 11), you will need to load a total of 400 μl of original cell culture supernatant into 2 separate Dialysis Tubes (200 μl /tube). Note: If the samples appear to be cloudy, transfer the samples to a clean tube, centrifuge at 13,000 rpm for 20 minutes at 2-8°C. If the samples are still not clear, store them at -20°C for 20 minutes. Remove from the freezer, immediately centrifuge at 13,000 rpm for 20 minutes at 2-8°C. 3. Place Floating Dialysis Rack into ≥500 ml dialysis buffer in a large beaker. Place beaker on a stir plate and dialyze, for at least 3 hours at 4°C, stirring buffer gently. Then exchange the 1X PBS buffer and repeat dialysis for at least 3 hours at 4°C. Transfer dialyzed sample to a clean microfuge tube. Spin dialyzed samples for 5 min at 10,000 rpm to remove any particulates or precipitates, and then transfer the supernatants to a clean tube. Note: The sample volume may change during dialysis. Note: Dialysis procedure may proceed overnight. Note: Determine the total protein concentration for cell culture supernatants or cell/tissue lysate after dialysis procedure (Step 3). We recommended using a BCA total protein assay (eg, Pierce, Catalog # 23227). B. Biotin-labeling Sample Note: Amines (e.g., Tris, glycine) and azides quench the biotinylation reaction. Avoid contaminating samples with these chemicals prior to biotinylation. 4. Immediately before use, prepare 1X Labeling Reagent. Briefly spin down the Labeling Reagent tube (Item B). Add 100 µl 1X PBS into the tube, then pipette up and down or vortex slightly to dissolve the lyophilized reagent. 5. Add 1X Labeling Reagent to dialyzed samples. RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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a. For labeling cell culture supernatants: transfer 180 μl dialyzed sample into a new tube. Add 36 μl of 1X Labeling Reagent Solution per 1 mg total protein in dialyzed cell culture supernatant. Mix well. For example, if sample’s total protein concentration is 0.5 mg/ml you need to add 3.24 µl 1X Labeling Reagent to the tube of 180 μl dialyzed sample. Note: You need to biotin-label 360 μl of dialyzed sample if dilution of the biotin-labeled samples is 2 fold in step 11 on page 11. b. For labeling serum or plasma: Add 22 μl of 1X Labeling Reagent Solution into a new tube containing 35 μl dialyzed serum or plasma sample and 155 μl Labeling Buffer (Item K). c. For labeling cell or tissue lysates: transfer 30 µg (15 μl of 2 mg/ml) cell or tissue lysates into a tube and add labeling buffer (Item K) for a total volume of 260 μl. Then add 3.3 μl of 1X Labeling Reagent Solution. Note: To normalize serum/plasma or cell/tissue lysate concentrations during biotinylation, measure sample volume before and after dialysis. Then adjust the volumes of dialyzed serum/plasma or cell/tissue lysates and Labeling Buffer to compensate. For example, if the sample volume doubles after dialysis, then use twice as much serum/plasma in the labeling reaction (70 μl) and reduce the Labeling Buffer to 120 μl. 6. Incubate the reaction solution at RT with gentle rocking or shaking for 30 min. Mix the reaction solution by gently tapping the tube every 5 minutes. 7. Add 3 μl Stop Solution (Item D) into each reaction tube and immediately dialyze as directed in Steps 1–3 on pages 8-9. Note: Biotinylated samples can be stored at -20°C or -80°C until you are ready to proceed with the assay.

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C. Drying the Glass Slide 8. Remove the package containing the Assembled Glass Slide (Item E) from the freezer. Place unopened package on the bench top for approx. 15 min, and allow the Assembled Glass Slide to equilibrate to RT. 9. Open package, and take the Assembled Glass Slide out of the sleeve (Do not disassemble the Glass Slide from the chamber assembly). Place glass slide assembly in laminar flow hood or similar clean environment for 1-2 hours at RT. Note: Protect the slide from dust or other contaminants. D. Blocking and Incubations Note: Glass slide should be completely dry before adding Blocking Buffer to wells. 10. Block sub-arrays by adding 400 μl of Blocking Buffer (Item F) into each well of Assembled Glass Slide and incubating at RT for 30 min. Ensure there are no bubbles on the array surfaces. 11. Immediately prior to sample incubation, spin biotin-labeled samples for 5 min at 10,000 rpm to remove any particulates or precipitates. Dilute samples with Blocking Buffer. Recommended dilution of the biotin-labeled samples with Blocking Buffer is 2-10 fold for cell culture supernatants, 20-fold for serum/plasma and 30 fold cell/tissue lysate. Note: Optimal sample dilution factor will depend on the abundance of target proteins. If the background or antigen-specific antibody signals are too strong, the sample can be diluted further in subsequent experiments. If the signal is too weak, more concentrated samples can be used.

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12. Completely remove Blocking Buffer from each well. Add 400 μl of diluted samples into appropriate wells. Remove any bubbles on array surfaces. Incubate arrays with gentle rocking or shaking for 2 hours at RT or overnight at 4°C. Note: Avoid the flow of sample into neighboring wells. 13. Based on number of samples and remaining protocol, calculate the amount of 1X Wash Buffer I and 1X Wash Buffer II needed to complete the experiment. Separately dilute the required amounts of 20X Wash Buffer I Concentrate (Item G) 20-fold and 20X Wash Buffer II Concentrate (Item H) with ddH2O. 14. Decant the samples from each well, and wash 3 times with 800 μl of 1X Wash Buffer I at RT with gentle rocking or shaking for 5 min per wash. 15. Obtain a clean container (e.g., pipette tip box or slide-staining jar), place the Assembled Glass Slide into the container with enough volume of 1X Wash Buffer I to completely cover the entire assembly, and remove any bubbles in wells. Wash 2 times at RT with gentle rocking or shaking for 10 min per wash. 16. Decant the Wash Buffer I from each well, place the Assembled Glass Slide into the container with enough volume of 1X Wash Buffer II to completely cover the entire assembly, and remove any bubbles in wells. Wash 2 times at RT with gentle rocking or shaking for 5 min per wash. 17. Prepare 1X Cy3-Conjugated Streptavidin: a) Briefly spin down tube containing the Cy3-Conjugated Streptavidin (Item I) immediately before use. b) Add 1000 μl of Blocking Buffer into the tube to prepare a concentrated Cy3-Conjugated Streptavidin stock solution. Pipette up and down to mix gently (do not store the stock solution for later use). RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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c) To prepare 1X Cy3-Conjugated Streptavidin add 200 μl of the concentrated Cy3-Conjugated Streptavidin stock solution into a tube with 800 μl of Blocking Buffer. Mix gently. 18. Carefully remove Assembled Glass Slide from container. Remove all of Wash Buffer II from the wells. Add 400 μl of 1X Cy3-Conjugated Streptavidin to each sub-array. Cover the incubation chamber with the plastic adhesive strips. Note: Avoid exposure to light in Steps 19–25 by covering the Glass Slide Assembly with aluminum foil or incubate in a dark room. 19. Incubate with 1X Cy3-Conjugated Streptavidin at RT for 2 hours with gentle rocking or shaking. Note: Incubation may be done overnight at 4°C. 20. Decant the solution and disassemble the glass slide from the incubation frame and chamber. Disassemble the device by pushing clips outward from the side, as shown below. Carefully remove the glass slide from the gasket. Note: Be careful not to touch the printed surface of the glass slide, which is on the same side as the barcode. 21. Gently place the glass slide into 30 ml Centrifuge Tube (Item M). Add enough 1X Wash Buffer I to cover the entire glass slide (about 30 ml). Wash with gentle rocking or shaking for 10 min. Remove the wash buffer. Repeat 2 times for a total of 3 washes. 22. Repeat step 20, this time with 1X Wash Buffer II. Repeat one time for a total of two washes for 5 min per wash. 23. Finally, wash the glass slide with 30 ml of ddH2O for 5 min. Remove glass slide and decant water from Centrifuge Tube. 24. Remove buffer droplets from the slide completely by one of the following ways: RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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• Put the glass slide into the Slide Washer/Dryer, and dry the glass slide by centrifuge at 1,000 rpm for 3 minutes without cap. • Or, dry the glass slide by a compressed N2 stream. • Or gently apply suction with a pipette to remove buffer droplets. Do not touch the array, only the sides. Note: Make sure the finished glass slide is completely dry before scanning or storage. E. Fluorescence Detection 25. You may proceed immediately to scanning or you may store the slide at -20 °C in the Centrifuge Tube provided or at RT to scan at a later time. Note: Please protect the finished glass slides from temperatures above RT and store them in the dark. Do not expose glass slide to strong light, such as sunlight or a UV lamp. Note: If you need to repeat any of the incubation steps after finishing the experiment, you must first re-assemble the glass slide into the incubation chamber by following the steps as described below. To avoid breaking the printed glass slide, you may first want to practice assembling the device with a blank glass slide. 1. 2. 3. 4.

Apply slide to incubation chamber barcode facing upward (image A). Gently snap one edge of a snap-on side (image B). Gently press other of side against lab bench and push in lengthwise direction (image C). Repeat with the other side (image D)

A

B

C

D

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V. Antibody Array Maps and Target Lists A. RayBio® Human Antibody Array L-507 Map 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

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395

395

396

396

397

397

398

398

399

399

400

400

401

401

402

402

403

403

404

404

405

405

406

406

407

407

408

408

409

409

410

410

411

411

412

412

413

413

414

414

415

415

416

416

417

417

418

418

419

419

420

420

421

421

422

422

423

423

424

424

425

425

426

426

427

427

428

428

429

429

430

430

431

431

432

432

433

433

434

434

435

435

436

436

437

437

438

438

439

439

440

440

441

441

442

442

443

443

44

444

445

445

446

446

447

447

448

448

449

449

450

450

451

451

452

452

453

453

454

454

455

455

456

456

457

457

458

458

459

459

460

460

461

461

462

462

463

463

464

464

465

465

466

466

467

467

468

468

469

469

470

470

471

471

472

472

473

473

474

474

475

475

476

476

477

477

478

478

479

479

480

480

481

481

482

482

483

483

484

484

485

485

486

486

487

487

488

488

489

489

490

490

491

491

492

492

493

493

494

494

495

495

496

496

497

497

498

498

499

499

500

500

501

501

502

502

503

503

504

504

505

505

506

506

507

507

508

508

509

509

510

510

511

511

512

512

513

513

514

514

515

515

Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

P-3c

P-3c

P-2c

P-2c

P-1c

P-1c

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

15

B. RayBio Human Antibody Array L-507 Target List Number

Name

Number

Name

Number

Name

Number

Name

Number

Name

1

Positive 1a

61

CCR7

121

Eotaxin-2 / MPIF-2

181

GFR alpha-2

241

IL-1 R6 / IL-1 Rrp2

2

Positive 2a

62

CCR8

122

Eotaxin-3 / CCL26

182

GFR alpha-3

242

IL-1 R8

3

Positive 3a

63

CCR9

123

Epiregulin

183

GFR alpha-4

243

IL-1 R9

4

neg

64

CD14

124

ErbB2

184

GITR / TNFRF18

244

IL-1 ra

5

6Ckine

65

CD27 / TNFRSF7

125

ErbB3

185

GITR Ligand / TNFSF18

245

IL-1 sRI

6

Activin A

66

CD30 / TNFRSF8

126

ErbB4

186

Glucagon

246

IL-1 sRII

7

Activin B

67

CD30 Ligand / TNFSF8

127

Erythropoietin

187

Glut1

247

IL-2

8

Activin C

68

CD40 / TNFRSF5

128

E-Selectin

188

Glut2

248

IL-2 R alpha

9

Activin RIA / ALK-2

69

CD40 Ligand / TNFSF5 /CD154

129

FADD

189

Glut3

249

IL-2 R beta /CD122

10

Activin RIB / ALK-4

70

CD 163

130

FAM3B

190

Glut5

250

IL-2 R gamma

11

Activin RII A/B

71

Cerberus 1

131

Fas / TNFRSF6

191

Glypican 3

251

IL-3

12

Activin RIIA

72

Chem R23

132

Fas Ligand

192

Glypican 5

252

IL-3 R alpha

13

Adiponectin / Acrp30

73

Chordin-Like 1

133

FGF Basic

193

GM-CSF

253

IL-4

14

AgRP

74

Chordin-Like 2

134

FGF-BP

194

GM-CSF R alpha

254

IL-4 R

15

ALCAM

75

CLC

135

FGF R3

195

Granzyme A

255

IL-5

16

Angiogenin

76

CNTF

136

FGF R4

196

GREMLIN

256

IL-5 R alpha

17

Angiopoietin-1

77

CNTF R alpha

137

FGF R5

197

GRO

257

IL-6

18

Angiopoietin-2

78

Coagulation Factor III / Tissue Factor

138

FGF-4

198

GRO-a

258

IL-6 R

19

Angiopoietin-4

79

CRIM 1

139

FGF-5

199

Growth Hormone (GH)

259

IL-7

20

Angiopoietin-like 1

80

Cripto-1

140

FGF-6

200

Growth Hormone R (GHR)

260

IL-7 R alpha

21

Angiopoietin-like 2

81

CRTH-2

141

FGF-7 / KGF

201

HB-EGF

261

IL-8

22

Angiopoietin-like Factor

82

Cryptic

142

FGF-8

202

HCC-4 / CCL16

262

IL-9

23

Angiostatin

83

Csk

143

FGF-9

203

HCR / CRAM-A/B

263

IL-10

24

APJ

84

CTACK / CCL27

144

FGF-10 / KGF-2

204

Hepassocin

264

IL-10 R alpha IL-10 R beta

25

APRIL

85

CTGF / CCN2

145

FGF-11

205

GLO-1

265

26

AR (Amphiregulin)

86

CTLA-4 /CD152

146

FGF-12

206

HGF

266

IL-11

27

Artemin

87

CV-2 / Crossveinless-2

147

FGF-13 1B

207

HGFR

267

IL-12 p40

28

Axl

88

CXCL14 / BRAK

148

FGF-16

208

HRG-alpha

268

IL-12 p70

29

B7-1 /CD80

89

CXCL16

149

FGF-17

209

HRG-beta 1

269

IL-12 R beta 1

30

BAFF R / TNFRSF13C

90

CXCR1 / IL-8 RA

150

FGF-18

210

HVEM / TNFRSF14

270

IL-12 R beta 2

31

BCMA / TNFRSF17

91

CXCR2 / IL-8 RB

151

FGF-19

211

I-309

271

IL-13

32

BD-1

92

CXCR3

152

FGF-20

212

ICAM-1

272

IL-13 R alpha 1

33

BDNF

93

CXCR4 (fusin)

153

FGF-21

213

ICAM-2

273

IL-13 R alpha 2

34

beta-Catenin

94

CXCR5 /BLR-1

154

FGF-23

214

ICAM-3 (CD50)

274

IL-15

35

BAX

95

CXCR6

155

FLRG

215

ICAM-5

275

IL-15 R alpha

36

beta-NGF

96

D6

156

Flt-3 Ligand

216

IFN-alpha / beta R1

276

IL-16

37

BIK

97

DAN

157

Follistatin

217

IFN-alpha / beta R2

277

IL-17

38

BLC / BCA-1 / CXCL13

98

DANCE

158

Follistatin-like 1

218

IFN-beta

278

IL-17B

39

BMP-2

99

DcR3 / TNFRSF6B

159

Fractalkine

219

IFN-gamma

279

IL-17B R

40

BMP-3

100

Decorin

160

Frizzled-1

220

IFN-gamma R1

280

IL-17C

41

BMP-3b / GDF-10

101

Dkk-1

161

Frizzled-3

221

IGFBP-1

281

IL-17D

42

BMP-4

102

Dkk-3

162

Frizzled-4

222

IGFBP-2

282

IL-17E

43

BMP-5

103

Dkk-4

163

Frizzled-5

223

IGFBP-3

283

IL-17F

44

BMP-6

104

DR3 / TNFRSF25

164

Frizzled-6

224

IGFBP-4

284

IL-17R

45

BMP-7

105

DR6 / TNFRSF21

165

Frizzled-7

225

IGFBP-6

285

IL-17RC

46

BMP-8

106

Dtk

166

Galectin-3

226

IGFBP-rp1 / IGFBP-7

286

Positive 1b

47

BMP-15

107

EDA-A2

167

GASP-1 / WFIKKNRP

227

IGF-I

287

Positive 2b

48

BMPR-IA / ALK-3

108

EDAR

168

GASP-2 / WFIKKN

228

IGF-I SR

288

Positive 3b

49

BMPR-IB / ALK-6

109

EDG-1

169

GCP-2 / CXCL6

229

IGF-II

289

neg

50

BMPR-II

110

EGF

170

GCSF

230

IGF-II R

290

IL-17RD

51

BTC

111

EGF R / ErbB1

171

G-CSF R / CD 114

231

IL-1 alpha

291

IL-18 BPa

52

Cardiotrophin-1 / CT-1

112

EG-VEGF / PK1

172

GDF1

232

IL-1 beta

292

IL-18 R alpha /IL-1 R5

53

CCL14 / HCC-1 / HCC-3

113

EMAP-II

173

GDF3

233

IL-1 F5 / FIL1delta

293

IL-18 R beta /AcPL

54

CCL28 / VIC

114

ENA-78

174

GDF5

234

IL-1 F6 / FIL1 epsilon

294

IL-19

55

CCR1

115

Endocan

175

GDF8

235

IL-1 F7 / FIL1 zeta

295

IL-20

56

CCR2

116

Endoglin / CD105

176

GDF9

236

IL-1 F8 / FIL1 eta

296

IL-20 R alpha

57

CCR3

117

Endostatin

177

GDF11

237

IL-1 F9 / IL-1 H1

297

IL-20 R beta

58

CCR4

118

Endothelin

178

GDF-15

238

IL-1 F10 / IL-1HY2

298

IL-21

59

CCR5

119

EN-RAGE

179

GDNF

239

IL-1 R3 / IL-1 R AcP

299

IL-21 R

60

CCR6

120

Eotaxin / CCL11

180

GFR alpha-1

240

IL-1 R4 / ST2

300

IL-22

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

16

RayBio Human Antibody Array L-507 Target List… continued Number

Name

Number

Name

Number

Name

Number

Name

301

IL-22 BP

361

MMP-2

421

RANK / TNFRSF11A

481

TMEFF1 / Tomoregulin-1

302

IL-22 R

362

MMP-3

422

RANTES

482

TMEFF2

303

IL-23

363

MMP-7

423

RELM beta

483

TNF-alpha

304

IL-23 R

364

MMP-8

424

RELT / TNFRSF19L

484

TNF-beta

305

IL-24

365

MMP-9

425

ROBO4

485

TNF RI / TNFRSF1A

306

IL-26

366

MMP-10

426

S100 A8/A9

486

TNF RII / TNFRSF1B

307

IL-27

367

MMP-11 /Stromelysin-3

427

S100A10

487

TRADD

308

IL-28A

368

MMP-12

428

SAA

488

TRAIL / TNFSF10

309

IL-29

369

MMP-13

429

SCF

489

TRAIL R1 / DR4 / TNFRSF10A

310

IL-31

370

MMP-14

430

SCF R /CD117

490

TRAIL R2 / DR5 / TNFRSF10B

311

IL-31 RA

371

MMP-15

431

SDF-1 / CXCL12

491

TRAIL R3 / TNFRSF10C

312

BACE-1

372

MMP-16 / MT3-MMP

432

sFRP-1

492

TRAIL R4 / TNFRSF10D

313

FACX

373

MMP-19

433

sFRP-3

493

TRANCE

314

Insulin

374

MMP-20

434

sFRP-4

494

TREM-1

315

Insulin R

375

MMP-24 / MT5-MMP

435

sgp130

495

TROY / TNFRSF19

316

Insulysin / IDE

376

MMP-25 / MT6-MMP

436

SIGIRR

496

TSG-6

317

IP-10

377

MSP alpha Chain

437

Siglec-5/CD170

497

TSLP R

318

I-TAC / CXCL11

378

Musk

438

Siglec-9

498

TWEAK / TNFSF12

319

Kininostatin / kininogen

379

NAP-2

439

SLPI

499

TWEAK R / TNFRSF12

320

Kremen-1

380

NCAM-1 / CD56

440

Smad 1

500

Ubiquitin+1

321

Kremen-2

381

Neuritin

441

Smad 4

501

uPA

322

Latent TGF-beta bp1

382

NeuroD1

442

Smad 5

502

uPAR

323

LBP

383

Neuropilin-2

443

Smad 7

503

Vasorin

324

Lck

384

Neurturin

444

Smad 8

504

VCAM-1 (CD106) VE-Cadherin

325

LECT2

385

NGF R

445

Prdx6

505

326

Lefty - A

386

Nidgen-1

446

Soggy-1

506

VEGF

327

Leptin (OB)

387

NOV / CCN3

447

Sonic Hedgehog (Shh N-terminal)

507

VEGF R2 (KDR)

328

Leptin R

388

NrCAM

448

SPARC

508

VEGF R3

329

LFA-1 alpha

389

NRG1 Isoform GGF2

449

Spinesin

509

VEGF-B

330

LIF

390

NRG2

450

TACI / TNFRSF13B

510

VEGF-C

331

LIF R alpha

391

NRG3

451

Tarc

511

VEGF-D

332

LIGHT / TNFSF14

392

NT-3

452

TCCR / WSX-1

512

VEGI / TNFSF15

333

Lipocalin-1

393

NT-4

453

TECK / CCL25

513

WIF-1

334

Lipocalin-2

394

Orexin A

454

TFPI

514

WISP-1 / CCN4

335

LRP-1

395

Orexin B

455

TGF-alpha

515

XEDAR

336

LRP-6

396

OSM

456

TGF-beta 1

516

Neg

337

L-Selectin (CD62L)

397

Osteoactivin / GPNMB

457

TGF-beta 2

517

Neg

338

Lymphotactin / XCL1

398

Osteocrin

458

TGF-beta 3

518

Neg

339

Lymphotoxin beta / TNFSF3

399

Osteoprotegerin / TNFRSF11B

459

TGF-beta 5

519

Neg

340

Lymphotoxin beta R / TNFRSF3

400

OX40 Ligand / TNFSF4

460

TGF-beta RI / ALK-5

520

Neg

341

MAC-1

401

PARC / CCL18

461

TGF-beta RII

521

Neg

342

MCP-1

402

PD-ECGF

462

Grb2

522

Neg

343

MCP-2

403

PDGF R alpha

463

TGF-beta RIII

523

P-3c

344

MCP-3

404

PDGF R beta

464

Thrombopoietin (TPO)

524

P-2c

345

MCP-4 / CCL13

405

PDGF-AA

465

TPX

525

P-1c

346

M-CSF

406

PDGF-AB

466

Thrombospondin-1

347

M-CSF R

407

PDGF-BB

467

Thrombospondin-2

348

MDC

408

PDGF-C

468

Thrombospondin-4

349

MFG-E8

409

PDGF-D

469

Thymopoietin

350

MFRP

410

PECAM-1 /CD31

470

Tie-1

351

MICA

411

Pentraxin3 / TSG-14

471

Tie-2

352

MIF

412

Persephin

472

TIMP-1

353

MIG

413

PF4 / CXCL4

473

TIMP-2

354

MIP-1a

414

PlGF

474

TIMP-3

355

MIP-1b

415

PLUNC

475

TIMP-4

356

MIP-1d

416

Pref-1

476

TL1A / TNFSF15

357

MIP 2

417

Progranulin

477

TLR1

358

MIP-3 alpha

418

Prolactin

478

TLR2

359

MIP-3 beta

419

P-selectin

479

TLR3

360

MMP-1

420

RAGE

480

TLR4

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

17

C. RayBio Human Antibody Array L-493 Map 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

P-1a

P-1a

P-2a

P-2a

P-3a

P-3a

Neg

Neg

5

5

6

6

7

7

8

8

9

9

10

10

11

11

12

12

13

13

14

14

15

15

16

16

17

17

18

18

19

19

20

20

21

21

22

22

23

23

24

24

25

25

26

26

27

27

28

28

29

29

30

30

31

31

32

32

33

33

34

34

35

35

36

36

37

37

38

38

39

39

40

40

41

41

42

42

43

43

44

44

45

45

46

46

47

47

48

48

49

49

50

50

51

51

52

52

53

53

54

54

55

55

56

56

57

57

58

58

59

59

60

60

61

61

62

62

63

63

64

64

65

65

66

66

67

67

68

68

69

69

70

70

71

71

72

72

73

73

74

74

75

75

76

76

77

77

78

78

79

79

80

80

81

81

82

82

83

83

84

84

85

85

86

86

87

87

88

88

89

89

90

90

91

91

92

92

93

93

94

94

95

95

96

96

97

97

98

98

99

99

100

100

101

101

102

102

103

103

104

104

105

105

106

106

107

107

108

108

109

109

110

110

111

111

112

112

113

113

114

114

115

115

116

116

117

117

118

118

119

119

120

120

121

121

122

122

123

123

124

124

125

125

126

126

127

127

128

128

129

129

130

130

131

131

132

132

133

133

134

134

135

135

136

136

137

137

138

138

139

139

140

140

141

141

142

142

143

143

144

144

145

145

146

146

147

147

148

148

149

149

150

150

151

151

152

152

153

153

154

154

155

155

156

156

157

157

158

158

159

159

160

160

161

161

162

162

163

163

164

164

165

165

166

166

167

167

168

168

169

169

170

170

171

171

172

172

173

173

174

174

175

175

176

176

177

177

178

178

179

179

180

180

181

181

182

182

183

183

184

184

185

185

186

186

187

187

188

188

189

189

190

190

191

191

192

192

193

193

194

194

195

195

196

196

197

197

198

198

199

199

200

200

201

201

202

202

203

203

204

204

205

205

206

206

207

207

208

208

209

209

210

210

211

211

212

212

213

213

214

214

215

215

216

216

217

217

218

218

219

219

220

220

221

221

222

222

223

223

224

224

225

225

226

226

227

227

228

228

229

229

230

230

231

231

232

232

233

233

234

234

235

235

236

236

237

237

238

238

239

239

240

240

241

241

242

242

243

243

244

244

245

245

246

246

247

247

248

248

249

249

250

250

251

251

252

252

253

253

254

254

255

255

256

256

257

257

258

258

259

259

260

260

261

261

262

262

263

263

264

264

265

265

266

266

267

267

268

268

269

269

270

270

271

271

272

272

273

273

274

274

275

275

276

276

277

277

278

278

279

279

280

280

281

281

282

282

283

283

284

284

285

285

P-1b

P-1b

P-2b

P-2b

P-3b

P-3b

Neg

Neg

290

290

291

291

292

292

293

293

294

294

295

295

296

296

297

297

298

298

299

299

300

300

301

301

302

302

303

303

304

304

305

305

306

306

307

307

308

308

309

309

310

310

311

311

312

312

313

313

314

314

315

315

316

316

317

317

318

318

319

319

320

320

321

321

322

322

323

323

324

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Neg

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Neg

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P-2c

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P-1c

P-1c

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D. RayBio Human Antibody Array L-493 Target List Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Name Positive-1a Positive-2a Positive-3a Blank 11b-HSD1 2B4 4-1BB ABL1 ACE ACE-2 ACK1 ACPP ACTH ADAM-9 Neurokinin-A ADAMTS-1 ADAMTS-L2 ADAMTS-4 ADAMTS-5 ADAMTS-10 ADAMTS-13 ADAMTS-15 ADAMTS-17 ADAMTS-18 ADAMTS-19 Adipsin Afamin AFP ALBUMIN IL-36RN Aldolase A Aldolase B Aldolase C ALK Alpha Lactalbumin Alpha 1 AG A1BG A1M A2M TPM1 ALPP pro-MMP13 AMICA AMPKa1 Amylin ANGPTL3 ANGPTL4 Annexin A7 APC APCS Apelin Apex1 APN ApoA1 ApoA2 ApoA4 ApoB ApoC2 ApoB100 ApoE

Number 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

Name ApoE3 ApoD ApoM ApoH APP ASPH Attractin B3GNT1 BAF57 BAFF BAI-1 BCAM Beta 2M Beta Defensin 4 Beta IG-H3 Biglycan BLAME BMP-9 BMX BNIP2 Btk ApoC1 CA 9 CA 15-3 CA 19-9 CA 125 Cadherin-13 Calbindin Calbindin D Calcitonin Calreticulin Calsyntenin-1 CPN2 CART Caspase-3 Caspase-8 Cathepsin B Cathepsin D Cathepsin L Cathepsin S CBP CCK CD23 CD24 CD36 CD38 CD44 CD45 CD46 CD47 CD55 CD59 CD71 CD74 CD90 CD97 CD 79 alpha CD200 CEA CEACAM-1

Number Name 121 Ceruloplasmin 122 CFHR2 123 Chemerin 124 CHI3L1 125 Chromogranin A 126 Chymase 127 cIAP-2 128 Ck beta 8-1 129 CK-MB 130 Claudin-3 131 Claudin-4 132 CLEC3B 133 Clusterin 134 CNDP1 135 Factor XIII A 136 Factor XIII B 137 COCO 138 C2 139 C3a 140 C5/C5a 141 C7 142 C8B 143 C9 144 Complement factor H 145 Contactin-1 146 Contactin-2 147 Corticosteroid-binding globulin 148 COX-2 149 C-peptide 150 Creatinine 151 CRP 152 CRTAM 153 CSH1 154 gamma-Thrombin 155 CutA 156 cTnT 157 Cyclin D1 158 Cystatin A 159 Cystatin B 160 Cystatin C 161 Cytochrome C 162 Cytokeratin 8 163 Cytokeratin 18 164 Cytokeratin 19 165 DBI 166 DCBLD2 167 D-Dimer 168 DEFA1/3 169 Defensin 170 Desmin 171 DLL1 172 DLL4 173 DMP-1 174 DPPIV 175 BNP 176 E-Cadherin 177 Endorphin Beta 178 Endothelin Receptor A 179 Enolase 2 180 ENPP2

Number 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

Name EpCAM EphA1 EphA2 EphA3 EphA4 EphA5 EphA6 EphA7 EphA8 EphB1 EphB2 EphB3 EphB4 EphB6 ERRa Erythropoietin R ESAM EV15L EXTL2 FABP1 FABP2 FABP4 FAK FAP Fc RIIB/C Fen 1 FER Ferritin Fetuin A Fetuin B FGFR1 FGFR1 alpha FGFR2 Fibrinogen Fibrinopeptide A Fibronectin Ficolin-3 FIH FOLR1 FOXN3 FoxO1 FoxP3 FRK FSH Furin Fyn GADD45A Galectin-1 Galectin-3BP Galectin-7 Gas1 Gastrin GATA-3 GATA-4 Gelsolin Ghrelin GLP-1 GPI GPBB GMNN

Number 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300

Name GPR-39 GPX1 GPX3 Pancreastatin GRP GRP75 GRP78 GSR GST HADHA HAI-1 HAI-2 hCG alpha hCGb Hck HE4 Hemopexin Hepcidin HSP32 HOXA10 Haptoglobin HSP10 HSP20 HSP27 HSP40 HSP60 HSP70 HSP90 HSPA8 HTRA2 IBSP IGF2BP1 IGFBP-5 IL-23p19 IL-33 IL-34 INSRR Integrin alpha V CD61 Itk ITM2B Kallikrein 2 ApoC3 Kallikrein 5 Kallikrein 6 Positive-1b Positive-2b Positive-3b neg Kallikrein 7 Kallikrein 8 Kallikrein 10 Kallikrein 11 Kallikrein 14 KCC3 KCTD10 KIF3B KLF4 LAG-3 pro-Glucagon

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RayBio® Human Antibody Array L-493 Target List ...continued Number 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

Name Layilin LDL R Legumain LH LIMPII LIN41 Livin LOX-1 LPS LRG1 LTF LTK Lumican Lyn LYRIC LYVE-1 LZTS1 Mammaglobin A Marapsin MATK MBL MBL-2 Mer Mesothelin MICB Midkine MINA FABP3 MSHa MTUS1 Myoglobin NAIP Nanog NELL2 NEP Galanin Nesfatin Nestin NET1 Netrin G2 Netrin-4 Neuropeptide Y NF1 NM23-H1/H2 Presenilin 2 Notch-1 NPTX1 NPTXR Progesterone Ntn1 OCT3/4 Omentin Osteocalcin Osteopontin OX40 p21 p27 p53 PAI-1 PAK7

Number 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

Name Pappalysin-1 Pancreatic Polypeptide Presenilin 1 PARK7 Visfatin P-Cadherin PCAF PD-1 PTH Troponin C PDX-1 PEDF PEPSINOGEN I PEPSINOGEN II Vasopressin PGRP-S PI 16 PI 3Kinase p85 beta PIM2 PKM2 Plasminogen Podocalyxin POMC PON1 PON2 PPARg2 PPP2R5C NR3C3 INSL3 Pro-BDNF Procalcitonin Pro-Cathepsin B Thrombin Prohibitin ProSAAS Prostasin PSP Pro-MMP-7 Pro-MMP-9 Protein p65 PSA-Free PSA-total PTHLP PTN PTPRD PYK2 PYY Ras RBP4 RECK RELM alpha Resistin RET RIP1 ROCK1 ROCK2 ROR1 ROR2 ROS RYK

Number 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480

Name S100A4 S100A6 S100A8 S-100b SART1 SART3 SCG3 Selenoprotein P SEMA3A Serotonin Serpin A1 Serpin A12 Serpin A3 Serpin A4 Serpin A5 Serpin A8 Serpin A9 Serpin B5 Serpin D1 Serpin I1 SERPING1 SERTAD2 SHBG SMAC SNCG SSTR5 Somatotropin SOST SOX17 SOX2 SPARCL1 SPINK1 SRMS SSEA-1 SSEA-4 SSTR2 Survivin SYK Syndecan-1 Syndecan-3 TACE TAF4 Tyk2 Tec TFF3 Thrombomodulin Thymidine Kinase-1 Thyroglobulin TIM-1 TNK1 TOPORS TPA TRA-1-60 TRA-1-81 Transferrin Trappin-2 TRKB TROPONIN I TYRO10 TRPC1

Number 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

Name TRPC6 TRPM7 Trypsin 1 TSH TSLP TXK Uromodulin TFF1 VDUP-1 VEGF R1 VGF VIP Receptor 2 Vitamin D Receptor Vitamin D-BP Vitamin K-dependent protein S Vitronectin VWF Wilms Tumor 1 XIAP ZAG ZAP70 Neg Neg Neg Neg Neg Neg Positive-3c Positive-2c Positive-1c

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VI. Interpretation of Results: A. Explanation of Controls Spots 1)

Positive Control spots (POS1, POS2, POS3) are standardized amounts of biotinylated IgGs printed directly onto the array. All other variables being equal, the Positive Control intensities will be the same for each sub-array. This allows for normalization based upon the relative fluorescence signal responses to a known control, much as “housekeeping” genes or proteins are used to normalize results in PCR or Western blots, respectively.

2)

Negative Control (NEG) spots contain a protein-containing buffer (used to dilute antibodies printed on the array). Their signal intensities represent non-specific binding of the Cy3-Conjugated Streptavidin. Negative control signal intensities are usually very close to background signals in each sub-array.

B. Typical Results The following figure shows the RayBio® L-Series Human Antibody Array 1000 probed with serum sample. The images were captured using a Axon GenePix laser scanner. The strong signals in row 20 and the upper left and lower right corners of each array are Positive Controls, which can be used to identify the orientation and help normalize the results between arrays.

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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RayBio® L-Series Human Antibody Array 507

Sample-1

Sample-2

RayBio® L-Series Human Antibody Array L-493

Sample-1

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

Sample-2

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If scanned using optimal settings, 3 distinct signal intensities will be seen: POS1>POS2>POS3. If all of these signals are of similar intensity, try increasing or decreasing laser power and/or signal gain settings. Note: In the absence of an external standard curve for each protein detected, there is no means of assessing absolute or relative concentrations of different proteins in the same sample using immunoassays. If you wish to obtain quantitative data (ie, concentrations of the various analytes in your samples), try using our Quantibody® Arrays as a targeted follow up experiment. C. Background Subtraction Once you have obtained fluorescence intensity data, you should subtract the background and normalize to the Positive Control signals before proceeding to analysis. Most laser fluorescence scanners’ software have an option to automatically measure the local background around each spot. For best results, we recommend comparing signal intensities representing the MEDIAN background signals minus local background. If your resulting fluorescence signal intensity reports do not include these values (e.g., a column labeled as “MED532-B532”), you may need to subtract the background manually or change the default settings on your scanner’s data report menu. D. Normalization of Array Data To normalize signal intensity data, one sub-array is defined as "reference" to which the other arrays are normalized. This choice is arbitrary. For example, in our Analysis Tool Software (described below), the array represented by data entered in the left-most column each worksheet is the default “reference array.”

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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You can calculate the normalized values as follows: X(Ny) = X(y) * P1/P(y) Where: P1 = mean signal intensity of POS spots on reference array P(y) = mean signal intensity of POS spots on Array "y" X(y) = mean signal intensity for spot "X" on Array "y" X(Ny) = normalized signal intensity for spot "X" on Array "y" The RayBio® Analysis Tool software is available for use with data obtained using RayBio® Biotin Label-based Antibody Arrays. You can copy and paste your signal intensity data (with and without background) into the Analysis Tool, and it will automatically normalize signal intensities to the Positive Controls. To order the Analysis Tool, please contact us at +1-770-729-2992 or [email protected] for more information. E. Threshold of Significant Difference After subtracting background signals and normalization to Positive Controls, comparison of signal intensities between and among array images can be used to determine relative differences in expression levels of each protein between samples or groups. Any ≥1.5-fold increase or ≤0.65-fold decrease in signal intensity for a single analyte between samples or groups may be considered a measurable and significant difference in expression, provided that both sets of signals are well above background (Mean background + 2 standard deviations, accuracy ≈ 95%).

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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VII. Troubleshooting Guide Problem

Cause Inadequate detection Inadequate reagent volumes or improper dilution Short incubation time

Weak Signal Too low protein concentration in sample Improper storage of kit Bubble formed during incubation Uneven signal

Arrays are not completed covered by reagent Reagent evaporation Cross-contamination from neighboring wells Comet tail formation

General

Inadequate detection

Overexposure Dark spots High background

Insufficient wash Dust Slide is allowed to dry out

Recommendation Increase laser power and PMT parameters Check pipettes and ensure correct preparation Ensure sufficient incubation time and change sample incubation step to overnight Dilute starting sample less or concentrate sample Store kit as suggested temperature. Don’t freeze/thaw the slide. Handle and pipette solutions more gently; De-gas solutions prior to use Prepare more reagent and completely cover arrays with solution Cover the incubation chamber with adhesive film during incubation Avoid overflowing wash buffer between wells Air dry the slide for at least 1 hour before usage Increase laser power so the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated Lower the laser power Completely remove wash buffer in each wash step Increase wash time and use more wash buffer Minimize dust in work environment before starting experiment Take additional precautions to prevent slides from dying out during experiment

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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VIII. Selected References Christina Scheel et all., Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast. Cell. 2011;145, 926–940 Lin Y, Huang R, Chen L, et al., Profiling of cytokine expression by biotinlabeled-based protein arrays. Proteomics. 2003, 3: 1750–1757. Huang R, Jiang W, Yang J, et al., A Biotin Label-based Antibody Array for High-content Profiling of Protein Expression. Cancer Genomics Proteomics. 2010; 7(3):129–141. Liu T, Xue R, Dong L, et al., Rapid determination of serological cytokine biomarkers for hepatitis B–virus-related hepatocellulare carcinoma using antibody arrays. Acta Biochim Biophys Sin. 2011; 43(1):45–51. Cui J, Chen Y, Chou W-C, et al., An integrated transcriptomic and computational analysis for biomarker identification in gastric cancer. Nucl Acids Res. 2011; 39(4):1197–1207. Jun Zhong et all., Temporal Profiling of the Secretome during Adipogenesis in Humans. Journal of Proteome Research. 2010, 9, 5228–5238 Chowdury UR, Madden BJ, Charlesworth MC, Fautsch MP., Proteomic Analysis of Human Aqueous Humor. Invest Ophthalmol Visual Sci. 2010; 51(10):4921–4931. Wei Y, Cui C, Lainscak M, et al., Type-specific dysregulation of matrix metalloproteinases and their tissue inhibitors in end-stage heart failure patients: relationshp between MMP-10 andLV remodeling. J Cell Mol Med. 2011; 15(4):773–782. Kuranda K, Berthon C, Lepêtre F, et al., Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem/progenitorlike state. J Cell Biochem. 2011; 112(5):1277–1285. Toh HC, Wang W-W, Chia WK, et al., Clinical Benefit of Allogenic Melanoma Cell Lysate-Pulsed Autologous Dendritic Cell Vaccine in MAGEPositive Colorectal Cancer Patients. Clin Chem Res. 2009; 15:7726–7736. RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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Zhen Hou, Cytokine array analysis of peritoneal fluid between women with endometriosis of different stages and those without endometriosi. Biomarkers. 2009;14(8): 604-618. Yao Liang Tang, et al., Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4. Circ Res. 2009;109:197723

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RayBio® L-series Antibody Arrays are patent-pending technology developed by RayBiotech. This product is intended for research only and is not to be used for clinical diagnosis. Our produces may not be resold, modified for resale, or used to manufacture commercial products without written approval by RayBiotech, Inc. Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials. Products are guaranteed for six months from the date of shipment when handled and stored properly. In the event of any defect in quality or merchantability, RayBiotech’s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price.

RayBio® is a registered trademark of RayBiotech, Inc. HyLite Plus™ is a trademark of Anaspec, Inc. GenePix® is a registered trademark of Molecular Devices, Inc.

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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This product is for research use only.

©2011 RayBiotech, Inc.

RayBio® L-Series Human Antibody Array L-507 or L-493 Protocol

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